Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo

Abstract

Establishment and maintenance of differential chromatin structure between transcriptionally competent and repressed genes are critical aspects of transcriptional regulation. The elements and mechanisms that mediate formation and maintenance of these chromatin states in vivo are not well understood. To examine the role of the promoter in maintaining chromatin structure and DNA methylation patterns of the transcriptionally active X-linked HPRT locus, 323 bp of the endogenous human HPRT promoter (from position ؊222 to ؉102 relative to the translation start site) was replaced by plasmid sequences by homologous recombination in cultured HT-1080 male fibrosarcoma cells. The targeted cells, which showed no detectable HPRT transcription, were then assayed for effects on DNase I hypersensitivity, general DNase I sensitivity, and DNA methylation patterns across the HPRT locus. In cells carrying the deletion, significantly diminished DNase I hypersensitivity in the 5 flanking region was observed compared to that in parental HT-1080 cells. However, general DNase I sensitivity and DNA methylation patterns were found to be very similar in the mutated cells and in the parental cells. These findings suggest that the promoter and active transcription play a relatively limited role in maintaining transcriptionally potentiated epigenetic states.An epigenetic hallmark of transcriptionally competent loci is that they adopt and maintain an open and accessible chromatin conformation, whereas inactive genes form a closed, inaccessible conformation (46). As assayed by sensitivity to digestion by nucleases such as DNase I, transcriptionally competent chromatin is characterized by generalized accessibility and sensitivity to DNase I digestion relative to bulk (as well as transcriptionally repressed) chromatin in vivo. This general DNase I sensitivity occurs along the entire length of an active gene and commonly extends both upstream and downstream of the gene itself (20,23,29). The difference in general DNase I sensitivity between transcriptionally competent chromatin and repressed chromatin is thought to reflect a difference in higher-order chromatin structure (43), though the molecular basis of general DNase I sensitivity and the resistance of chromatin are not well understood.DNase I-hypersensitive regions of chromatin in transcriptionally active loci are commonly associated with cis-acting regulatory elements and are thought to reflect localized disruption of nucleosomal arrays (17). This localized hypersensitivity to DNase I is commonly believed to result from the binding of transcription factors to their cognate recognition sequences, leading to alteration of local nucleosomal organization and/or structure (2, 16). However, several studies also suggest that formation and/or maintenance of DNase I-hypersensitive sites in the 5Ј flanking regions of active genes may arise independent of stable transcription factor binding in the promoter (10,25,28,31,33).Mechanisms that establish and maintain transcriptionally competent or repressed ch...

Regulatory Elements Associated with Paternally-Expressed Genes in the Imprinted Murine Angelman/Prader-Willi Syndrome Domain

“…Analysis of DNase I hypersensitivity by Southern blot analysis was performed as described previously [17], [25]. For analysis of primary mouse brain cells, cell suspensions were prepared from one day old newborn mice as described above, permeablized cells were treated with increasing concentrations of DNase I (Worthington) in suspension, then purified genomic DNA from each sample was digested with Eco RI, Taq I, or Nco I, and Southern blotted.…”

“…For analysis of primary mouse brain cells, cell suspensions were prepared from one day old newborn mice as described above, permeablized cells were treated with increasing concentrations of DNase I (Worthington) in suspension, then purified genomic DNA from each sample was digested with Eco RI, Taq I, or Nco I, and Southern blotted. Hybridization probes for Southern blot analyses were isolated and radiolabeled by random priming as described previously [17], [25]. Hybridization probe A was a 475 bp fragment spanning positions +1397 to +1872 (relative to the transcription initiation site) of the Snrpn gene synthesized by PCR and cloned.…”

Locus control region elements HS2 and HS3 in combination with chromatin boundaries confer high‐level expression of a human β‐globin transgene in a centromeric region

“…Cells taken from a spleen of mice made anaemic by phenylhydrazine injection (see RNA isolation section) were washed with PBS, pelleted and subjected to DNase I digestion as described by Kang et al . (2003).…”

“…Cells taken from a spleen of mice made anaemic by phenylhydrazine injection (see RNA isolation section) were washed with PBS, pelleted and subjected to DNase I digestion as described by Kang et al (2003). DNase I (10 µg)-treated DNA was digested with EcoRI, size fractionated on a 0.8% agarose gel and subjected to Southern blot analysis using the β-mid probe as described above.…”

Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins

“…Utilizing a recently developed quantitative long-range PCR-based assay for measuring genespecific removal of UV-induced lesions [7], we assessed the efficiency of removal of UVinduced DNA damage in two different human genes: the hypoxanthine phosphoribosyl transferase (HPRT) gene, an allele of which is constitutively expressed in all normal somatic cells [8][9][10]), and the β-globin gene, which is transcriptionally silent in all cells but those of erythroid lineage [11]. These genes were chosen because microarray and other analyses indicated that HMGA1 proteins do not regulate the transcription of either gene in the cell lines being investigated (unpublished data), an important consideration given that these proteins can either positively or negatively regulate the transcription of a large number of genes in vivo (reviewed in: [12]).…”